Identification of the major photodegradant in metronidazole by LC-PDA-MS and its reveal in compendial methods

Metronidazole in aqueous solution is sensitive to light and UV irradiation, leading to the formation of N-(2-hydroxyethyl)-5-methyl-l,2,4-oxadiazole-3-carboxamide. This is revealed here by liquid chromatography with tandem photo diode array detection and mass spectrometry (LC-PDA-MS) and further verified by comparison with the corresponding reference substance and proton nuclear magnetic resonance (1H-NMR). However, in current compendial tests for related substances/organic impurities of metronidazole, the above photolytic degradant could not be detected. Thus, when photodegradation of metronidazole occurs, it could not be demonstrated. In our study, an improved LC method was developed and validated, which includes a detection at a wavelength of 230 nm and optimization of mobile phase composition thereby a better separation was obtained.

Unfortunately, in current compendial tests for related substances/organic impurities of metronidazole, the above photolytic degradant can not be detected at the prescribed wavelength (315/319 nm) [7][8][9][10][11] . Thus, when photodegradation of metronidazole occurs, it could not be demonstrated. Up till now, no paper has been published in literature describing a control method for the photodegradation product of metronidazole.
Analysis of light stressed metronidazole solutions revealed a mass imbalance leading to the start of this investigation. Indeed, degradation percentages of metronidazole were much higher than those explained by the degradants detected by current compendial methods.
For the structural characterization of the photolytic degradant of metronidazole, liquid chromatography with tandem mass spectrometry (LC-MS) was applied at first stage due to its high selectivity and sensitivity in the qualification of unknown compounds [12][13][14][15][16][17][18] . Further structural confirmation was performed by comparison with the corresponding reference substance (RS) in terms of retention time and extracted photo diode array (PDA) spectra of the peaks obtained in the chromatograms. Finally, proton nuclear magnetic resonance ( 1 H-NMR) of the photolytic degradant in CDCl 3 and D 2 O was performed because of its unambiguous chemical characterization. Light stressed samples of metronidazole drug substance in water (0.2 mg/mL) and its vaginal lotion (containing 10 mg metronidazole and 60 mg chlorhexidine gluconate in 50 mL, with as excipients: polysorbate 80 (1 mg/mL), ethanol (1.0%, v/v) and water) were obtained under UV irradiation of 5000 lx in one Labonce ® 500 TPS stability chamber (Beijing, P.R.China) at 25 °C for 30 days.
One sample of metronidazole injection (0.1 g/20 mL) from Wuhan Fuxing Biopharmaceutical Co. (Wuhan, Hubei, P.R. China), and samples of metronidazole with sodium chloride for injection from Shijiazhuang No.4 Pharmaceutical Co. (Shijiazhuang, Hebei, P.R. China) (0.5 g of metronidazole and 0.8 g of sodium chloride per 100 mL) and Shandong Qidu Pharmaceutical Co. (Jinan, Shandong, P.R. China) (0.5 g of metronidazole and 0.9 g of sodium chloride per 100 mL) were purchased from the Chinese market (Beijing, P.R. China). The above metronidazole samples for injection were treated under UV irradiation of 5000 lx in one Labonce ® 500 TPS stability chamber (Beijing, P.R.China) at 25 °C for 2 and 5 days, as well as at room light (close to the window) for 48 h, to demonstrate the photodegradation risk of metronidazole injection during administration over several hours 21 .

LC-PDA experiments.
In the stress study of metronidazole in aqueous solutions and the vaginal lotion, as well as in further verification testing of its major photolytic degradant, the LC system (Shimadzu, Darul Khusus, Malaysia) consisted of a binary pump (LC-2030C plus), an autosampler (LC-2030C plus), a photo diode array (PDA) detector (LC-2030C plus) and a LC-2030C plus column oven. Data acquisition, analysis and reporting were performed using Shimadzu LC-Solution software. The starting chromatographic conditions chosen were based on available compendial monographs of metronidazole [7][8][9][10][11] . The Kromasil 100-5 C 18 column (250 mm × 4.6 mm i.d., 5 μm) (AkzoNobel, Bohus, Sweden) was maintained at 30 °C. Mobile phase A (0.05 mol/L KH 2 PO 4 in water) and mobile phase B (methanol) were pumped at a total flow rate of 1.

Results
Method development and validation. In the metronidazole monograph of EP10 7 and BP2017 8 , a mobile phase is prescribed consisting of 1.36 g/L KH 2 PO 4 -methanol (70:30 v/v), while in USP43 9 and ChP2020 11 water-methanol (80:20 v/v) is prescribed. It was found in this study that a composition of 0.05 mol/L KH 2 PO 4methanol (80:20 v/v), as initial mobile phase showed a more ideal peak symmetry and better resolution between the peaks due to the photolytic degradant and its neighbouring impurities. Then, gradient elution was introduced for elution of chlorhexidine gluconate and its related substances. The proposed method (see "LC-PDA experiments" section for details) has been validated at a wavelength of 230 nm for its intended use in terms of specificity, sensitivity, accuracy, linearity, precision and robustness (see supplementary materials for details). The photolytic degradant was stable in the proposed methanol-water (20:80, v/v) solvent for at least 33 h at room temperature. No interferences from samples subjected to heat, acid, base or oxidation, known specified impurity (2-methtyl-5-nitro imidazole) or sample matrix were observed. The sensitivity (quantitation limit: 0.05%) met the intended identification threshold of 0.2% 24 Fig. 2), its corresponding UV spectrum (see Fig. 3, with poor absorption at 315 nm) and related references 1-6 , it was identified as N-(2-hydroxy ethyl)-5-methyl-l,2,4-oxadiazole-3-carboxamide (CAS:110578-73-9) , whose production pathway was proposed by rearrangement of metronidazole as described in Fig. 1 1-3 .

Photodegradation risk evaluation of metronidazole injection.
Immediately after opening the packaging, the above photolytic degradant amounted 0.01-0.02% in the two batches of metronidazole with sodium chloride for injection, while it could not be detected in the metronidazole injection without sodium chloride (detection limit: ~ 0.01%). However, its content increased to 0.15% at room light condition for 48 h, and to 0.36% and 0.88% respectively after 2 and 5 days under UV irradation (5000 lx), see supplementary materials for details. These were all higher than its identification threshold and the qualification threshold (maximum daily dose of metronidazole injection: 4 g) 21,24 , demonstrating the necessity to control the photolytic degradant in metronidazole injection, which is typically administered by slow intravenous drip infusion. Unfortunately, the instruction leaflet of marketed metronidazole injections only warned to protect from light during storage while their containers were not light-resistant and no light-protection precautions were proposed during administration 21 .
Suggestions to improve current compendial methods for related substances of metronidazole and its drug products. Based on the above information, it was proposed to modify compendial methods for related substances of metronidazole and its drug products, so the method could be used to monitor the specified impurities and the new photolytic degradant. The proposed method, as described in "LC-PDA experiments" section, has been fully validated for its intended use. The relative correction factor (F) of the photolytic degradant to metronidazole measured at 230 nm is about 0.9, within the acceptable range of 1.0 (0.8-1.2). The major photolytic degradant as well as other minor light stressed degradants could be observed with good sensitivity when the detection wavelength was switched from to 315 to 230 nm, as shown in Fig. 4.

Discussion
(1) Though photosensitization potentials are all emphasized in its storage and solution preparations of metronidazole in EP10, BP2017, USP43, JP17 as well as ChP2020, and real photodegradation was observed in commercial metronidazole injections, especially during its slow intravenous drip infusion, the photolytic degradant could not be detected by current compendial methods for related substances of metronidazole and its products at the described 315/319 nm wavelength. The observation revealed possible reason for the mass imbalance of light stressed metronidazole in solution and its vaginal lotion detected by current compendial methods for its related substances. Thus, suggestions to modify current compendial methods to monitor the new degradant were proposed, e.g. detection at an additional wavelength of 230 nm and modification of the gradient. And other possible precautions against light during its slow intravenous drip infusion of metronidazole injections or similar aqueous products were also suggested. The proposed method has also been fully validated, and its proposed limit (0.2%, maximum daily dose of metronidazole vaginal lotion: 20 mg) was based on ICH Q3B 24 . Maybe further investigations, e.g. its toxicological and pharmacological data, are required to reveal if the proposed limit is scientific and reasonable.  . Its typical parent ions, characteristic product ions as well as extracted PDA spectrum were totally in agreement with those of references 1-6 . Further verification was performed by comparison with the corresponding reference substance, in terms of retention times and extracted PDA spectra under the same chromatographic condition (see "LC-PDA experiments" section), which were consistent with each other. The characteristic differences between the 1 H-NMR data of the photolytic degradant RS and metronidazole RS in CDCl 3 and D 2 O (see supplementary materials) were consistent with reports 1-6 and its proposed structure as shown in Fig. 1, which further ensured reliability of the above structural characterization results of the degradant in light stressed metronidazole samples. If necessary, conducting photostress of metronidazole on large scale, then isolation/purification of the degradant maybe are required.